WO2005067960A1 - Peptides synthetiques derives de la hr2 de la gp41 du vih, et leur utilisation dans une therapie destinee a inhiber la transmission du virus de l'immunodeficience humaine - Google Patents

Peptides synthetiques derives de la hr2 de la gp41 du vih, et leur utilisation dans une therapie destinee a inhiber la transmission du virus de l'immunodeficience humaine Download PDF

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Publication number
WO2005067960A1
WO2005067960A1 PCT/US2004/042918 US2004042918W WO2005067960A1 WO 2005067960 A1 WO2005067960 A1 WO 2005067960A1 US 2004042918 W US2004042918 W US 2004042918W WO 2005067960 A1 WO2005067960 A1 WO 2005067960A1
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seq
synthetic peptide
hiv
base sequence
amino acid
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PCT/US2004/042918
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English (en)
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Mary K. Delmedico
John Dwyer
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Trimeris, Inc.
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Priority to EP04815041A priority Critical patent/EP1708734A4/fr
Priority to AU2004313242A priority patent/AU2004313242A1/en
Priority to BRPI0418304-5A priority patent/BRPI0418304A/pt
Priority to JP2006549305A priority patent/JP2007517522A/ja
Priority to CA002551082A priority patent/CA2551082A1/fr
Publication of WO2005067960A1 publication Critical patent/WO2005067960A1/fr
Priority to US11/477,574 priority patent/US20060247416A1/en
Priority to IL176721A priority patent/IL176721A0/en
Priority to NO20063556A priority patent/NO20063556L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to synthetic peptides derived from the HR2 region of
  • HIV Human Immunodeficiency Virus
  • the present invention comprises a family of peptides that contain a plurality of amino acid substitutions (as compared to the native sequence) which result in unexpected, improved biological activity.
  • gp120/gp41 interacts with cell surface receptors on the membranes of the target cells. Following binding of gp120 to cellular receptors (e.g., CD4 in combination with a chemokine co-receptor such as CCR-5 or CXCR-4), induced is a conformational change in the gp120/gp41 complex that allows gp41 to insert into the membrane of the target cell and mediate membrane fusion.
  • cellular receptors e.g., CD4 in combination with a chemokine co-receptor such as CCR-5 or CXCR-4
  • induced is a conformational change in the gp120/gp41 complex that allows gp41 to insert into the membrane of the target cell and mediate membrane fusion.
  • the amino acid sequence of gp41 and its variation among different strains of HIV, is well known.
  • FIG.1 is a schematic representation of the generally accepted functional domains of gp41 (note the amino acid sequence numbers may vary slightly depending on
  • the fusion peptide (fusogenic domain) is believed to be involved in insertion into and disruption of the target cell membrane.
  • the transmembrane domain containing the transmembrane anchor sequence, is located at the C-terminal end of the protein.
  • Between the fusion peptide and transmembrane anchor are two distinct regions, known as heptad repeat (HR) regions, each region having a plurality of heptads.
  • the HR1 region nearer to the N-terminal end of the protein than the HR2 region, has been generally described as comprising amino acid residues from about 545 to about 595 of the amino acid sequence of gp160. However, the amino acid numbering of gp160 depends on the strain from which the amino acid sequence was deduced.
  • the amino acid sequence comprising the HR1 region and the amino acid sequence comprising the HR2 region are each highly conserved regions in the HIV-1 envelope protein.
  • the HR2 region has been generally described as comprising amino acids in the positions from about 628 to about 678 of the amino acid sequence of gp160.
  • the HR regions have a plurality of 7 amino acid residue stretches or "heptads" (the 7 amino acids in each heptad designated "a” through "g"), wherein the amino acids in the "a" position and "d” position are generally hydrophobic.
  • each HR region is one or more leucine zipper-like motifs (also referred to as “leucine zipper-like repeats”) comprising an 8 amino acid sequence initiating with, and ending with, either an isoleucine or leucine.
  • leucine zipper-like repeats also referred to as "leucine zipper-like repeats”
  • the HR2 region has just one leucine zipper like-motif
  • the HR1 region has five leucine zipper-like motifs.
  • coiled coils are known to be comprised of two or more helices that wrap around each other in forming oligomers, with the hallmark of coiled coils being a heptad repeat of amino acids with a predominance of hydrophobic residues at the first ("a") and fourth ("d") positions, charged residues frequently at the fifth (“e") and seventh ("g") positions, and with the amino acids in the "a" position and "d” position being primary determinants that influence the oligomeric state and strand orientation. It was discovered that peptides derived from the native sequence of either the
  • HR1 region (“HR1 peptides”) or HR2 region (“HR2 peptides”) of HIV gp41 inhibit transmission of HIV to host cells both in in vitro assays and in in vivo clinical studies (see, e.g., Wild et al., 1994, Proc. Natl. Acad. Sci. USA, 91 :9770-9774; U.S. Patent Nos. 5,464,933 and 5,656,480 licensed to the present assignee; and Kilby et al., 1998, Nature Med. 4:1302-1306. See also, e.g., U.S. Patent Nos. 6,258,782 and 6,348,568 assigned to the present assignee.).
  • HR2 peptides as exemplified by DP178 (also known as T20, enfuvirtide, and Fuzeon® ; SEQ ID NO:1 ), T651 (SEQ ID NO:2), T649 (SEQ ID NO:3), blocked infection of target cells with potencies of 0.5 ng/ml (EC50 against HIV-1 LA) ; see, e.g., Lawless et al., 1996, Biochemistry, 35:13697-13708), 5 ng (IC50; HIV-1 IIIB). and 2 ng (IC50; HIV-1 IIIB), respectively.
  • the respective amino acid sequences of T651 (SEQ ID NO:2) and T649 (SEQ ID NO:3) are also disclosed in U.S.
  • Patent No. 6,479,055 (assigned to the present assignee). Further, clinical studies have shown that treatment of an HIV-infected individual with a regimen of antiviral agents containing T20 (SEQ ID NO: 1) significantly reduces the HIV-1 viral load, and significantly increases the circulating CD4 + cell population, in such treated individual as compared to that of an individual receiving the same regimen but without T20. Attempts have been made to improve the biological activity of HIV-derived HR2 peptides.
  • helix-favoring amino acid substitution i.e., ⁇ -aminoisobutyric acid
  • chemical crosslinkers i.e., a diaminoalkane crosslinker
  • Those peptides produced showed biological activity ranged only from about a 4 fold to a 15 fold increase in potency (e.g., inhibitory activity), whereas others showed no inhibitory activity (Sia et al., 2002, supra). Thus, only small gains in an already weak inhibitor (IC50 of >500 ⁇ M) were achieved by this method. Additionally, Sia et al. confirmed what is current thinking in the art; i.e., that, generally, there is a lack of correlation between helical propensity and biological activity. For example, peptides showing the highest helical content are often the weakest inhibitors of HIV- induced membrane fusion.
  • the biological activity of the 3 peptides showed either no increase or, at best, a 3 fold increase in inhibitory activity (EC50, anti-HIV activity using MAGI assays; Otaka et al., 2002, supra) as compared to the parent peptide into which the substitutions were introduced.
  • EC50 anti-HIV activity using MAGI assays
  • mutations can occur in HIV during treatment which reduce the sensitivity to drug therapy using fusion inhibitor peptides such as T20 (SEQ ID NO:1 ).
  • fusion inhibitor peptides such as T20 (SEQ ID NO:1 ).
  • such improved biological activity can include, but is not limited to, about a 100 fold to about a 1 ,000 fold increase in inhibitory activity (as compared to a peptide consisting of the base (native) HR2 sequence from which it was derived) against HIV strains having developed resistance to known HR2 peptides, and more particularly against HIV strains that have developed resistance to any one or more of the peptides represented by SEQ ID NOs: 2-4.
  • the present invention relates to synthetic peptides derived from a base amino acid sequence ("base sequence") of one or more of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, wherein the synthetic peptide differs from the base sequence by the addition of a plurality of amino acids, in replacing amino acids in the base sequence, wherein a synthetic peptide demonstrates an unexpected, improved biological activity, and may further comprise an increase in helicity, as compared to the base sequence from which it was derived.
  • each such synthetic peptide comprises a base sequence of one or more of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, but differs from the base sequence by further comprising a plurality of amino acid substitutions (as compared to the base sequence) comprising one or more helix-promoting amino acids, wherein the synthetic peptide demonstrates an unexpected, improved biological activity, and may further comprise an increase in helicity, as compared to the base sequence.
  • the plurality of substitutions with a helix-promoting amino acid can range from about 5 to about 15 amino acids, depending on the length of the base sequence from which it was derived; and more particularly from about 5% to about 50% of the amino acids of the base sequence may be replaced with one or more helix-promoting amino acids to produce a synthetic peptide according to the present invention.
  • a synthetic peptide comprising a base sequence of one or more of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, except that the synthetic peptide differs from the base sequence by: (a) a plurality of amino acid substitutions comprising one or more helix-promoting amino acids; (b) a plurality of charged amino acids which are spaced apart, in the amino acid sequence of the synthetic peptide, from oppositely charged amino acids in forming a plurality of ion pairs (preferably, either in an /, / + 4 arrangement and/or /,/ ' + 3 arrangement); and (c) demonstrating an improved biological activity.
  • the number of ion pairs in the synthetic peptide range from about 3 to about 10.
  • the synthetic peptide further comprises an increase in helicity, as compared to the base sequence from which it was derived.
  • the plurality of amino acid substitutions comprising one or more helix promoting amino acids and a plurality of charged amino acids can range from about 5 to about 25 amino acids, depending on the length of the base sequence from which it is derived; and more particularly, from about 5% to about 60% of the amino acids of the base sequence may be substituted with a combination of helix-promoting amino acids and charged amino acids to produce a synthetic peptide according to the present invention.
  • synthetic peptides derived from a base sequence of one or more of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, wherein the synthetic peptide has plurality of amino acid substitutions (as compared to the amino acid sequence of the base sequence from which it is derived) comprising one or more of: (a) a helix-promoting amino acid; and (b) a plurality of charged amino acids introduced to form ion pairs (preferably, in either an /,/ ' + 3 arrangement and/or /,/ ' + 4 arrangement) between oppositely charged amino acids; wherein the synthetic peptide has an improved biological activity.
  • the synthetic peptide may further comprise an increase in helicity, as compared to the base sequence from which it was derived, and has a stability as measured by a Tm (melting temperature) in the range of from about 36°C to about 75°C.
  • the plurality of amino acid substitutions comprising a helix-promoting amino acid and a charged amino acid can range from about 5 to about 25 amino acids, depending on the length of the base sequence; and more particularly from about 5% to about 60% of the amino acids of the base sequence may be substituted with a combination comprising a helix-promoting amino acid and a charged amino acid to produce a synthetic peptide according to the present invention.
  • the synthetic peptides according to the invention may further comprise an N- terminal group, a C-terminal group, or both an N-terminal group and C-terminal group, as described in more detail.
  • a synthetic peptide according to the present invention as an active therapeutic substance in therapy of HIV infection.
  • a synthetic peptide according to the present invention for the manufacture of a medicament for a therapeutic application comprising treatment of HIV.
  • a method for inhibition of transmission of HIV to a cell comprising contacting the virus in the presence of a cell with an amount of synthetic peptide according to the present invention effective to inhibit infection of the cell by HIV.
  • a method for inhibition of transmission of HIV to a cell comprising adding to the virus and the cell an amount of synthetic peptide effective to inhibit infection of the cell by HIV.
  • a method for inhibiting HIV fusion e.g., a process by which HIV gp41 mediates fusion between the viral membrane and cell membrane during infection by HIV of a target cell
  • HIV fusion comprising contacting the virus in the presence of a cell with a concentration of synthetic peptide according to the present invention effective to inhibit HIV membrane fusion.
  • FIG. 1 is a schematic of HIV gp41 showing the heptad repeat 1 region (HR1) and heptad repeat 2 region (HR2) along with other functional regions of gp41.
  • HR1 heptad repeat 1 region
  • HR2 heptad repeat 2 region
  • Exemplary peptide sequences corresponding to HIV LA ⁇ HR1 and HR2 are shown for purposes of illustration. The amino acid residues are numbered according to their position in gp160, strain HIVLM-
  • FIG. 2 shows a comparison of polymorphisms contained within SEQ ID NO:2 of the HR2 region of HIV gp41 as determined from various laboratory strains and clinical isolates, wherein variations in amino acid sequence are indicated by the single letter amino acid code.
  • the cell is a human cell or are human cells; and more preferably, human cells capable of being infected by HIV via a process including membrane fusion.
  • pharmaceutically acceptable carrier when used herein for purposes of the specification and claims, means a carrier medium that does not significantly alter the biological activity of the active ingredient (e.g., a synthetic peptide according to the present invention) to which it is added.
  • a suitable pharmaceutically acceptable carrier may comprise one or substances, including but not limited to, water, buffered water, saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer; and may further include one or more substances such as water-soluble polymer, glycerol, polyethylene glycol, glycerin, oils, salts such as sodium, potassium, magnesium and ammonium, phosphonates, carbonate esters, fatty acids, saccharides, polysaccharides, glycoproteins (for enhanced stability), excipients, and preservatives and/or stabilizers (to increase shelf-life or as necessary and suitable for manufacture and distribution of the composition).
  • the carrier is suitable for intravenous, intramuscular, subcutaneous or parenteral administration.
  • amino acid is meant, for purposes of the specification and claims and in reference to the synthetic peptides according to the present invention, to refer to a molecule that has at least one free amine group and at least one free carboxyl group.
  • the amino acid may have more than one free amine group, or more than one free carboxyl group, or may further comprise one or more free chemical reactive groups other than an amine or a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc.).
  • the amino acid may be a naturally occurring amino acid (e.g., L-amino acid), a non-naturally occurring amino acid (e.g., D-amino acid), a synthetic amino acid, a modified amino acid, an amino acid derivative, an amino acid precursor, and a conservative substitution.
  • L-amino acid a naturally occurring amino acid
  • D-amino acid a non-naturally occurring amino acid
  • synthetic amino acid a modified amino acid
  • an amino acid derivative e.g., an amino acid precursor
  • conservative substitution e.g., a conservative substitution.
  • One skilled in the art would know that the choice of amino acids incorporated into a peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the antiviral peptide. Such characteristics are determined, in part, by determination of helicity (as described herein in more detail) and antiviral activity (as described herein in more detail).
  • amino acids in a synthetic peptide may be comprised of one or more of naturally occurring (L)-amino acid and non-naturally occurring (D)-amino acid.
  • a preferred amino acid may be used to the exclusion of amino acids other than the preferred amino acid.
  • a "helix-promoting amino acid” is meant, for purposes of the specification and claims, to refer to an amino acid that has a high propensity to promote alpha helix formation of an amino acid sequence containing such helix-promoting amino acid.
  • Naturally occurring amino acids which are helix-promoting amino acids include glutamic acid, alanine, leucine, methionine, glutamine, isoleucine, lysine, arginine, phenylalanine, histidine, and trytophan, and non-naturally occurring amino acids such as an amino-butyric acid (e.g., ⁇ -aminoisobutyric acid).
  • the order of helical propensity is: glutamic acid, alanine, leucine, methionine, glutamine, lysine, arginine, phenylalanine, isoleucine, histidine, and trytophan.
  • a synthetic peptide comprises a plurality of amino acid substitutions, as compared to the base sequence from which it is derived, wherein an amino acid substitution comprises a helix-promoting amino acid, in a position in the amino acid sequence of the synthetic peptide, which has a higher helical propensity as compared to the amino acid in a corresponding position of amino acid sequence of the base sequence from which the synthetic peptide is derived.
  • an amino acid substitution comprises a helix-promoting amino acid, in a position in the amino acid sequence of the synthetic peptide, which has a higher helical propensity as compared to the amino acid in a corresponding position of amino acid sequence of the base sequence from which the synthetic peptide is derived.
  • a helix-promoting amino acid which is not charged is substituted for a charged amino acid in the base sequence.
  • helix-promoting when used herein for purposes of the specification and claims, is customarily referring to the effect of one or more amino acid substitutions on contributing to the helicity of a peptide; and more particularly, an effect observed as one or more of alpha helix stabilizing, or increase in helicity, as known in the art.
  • a "conservative substitution”, in relation to amino acid sequence of a synthetic peptide according to the present invention, is a term used hereinafter for the purposes of the specification and claims to mean one or more amino acids substitution in the sequence of the synthetic peptide such that the synthetic peptide still demonstrates (conserved is) the unexpected, improved biological activity, as described in more detail herein.
  • substitutions are defined by aforementioned function, and includes substitutions of amino acids having substantially the same charge, size, hydrophilicity, and/or aromaticity as the amino acid replaced.
  • substitutions are known to those of ordinary skill in the art to include, but are not limited to, glycine-alanine- valine; isoleucine-leucine; tryptophan-tyrosine; aspartic acid-glutamic acid; arginine- lysine; asparagine-glutamine; and serine-threonine.
  • substitutions may also comprise polymorphisms, as illustrated in FIG. 2, at the various amino acid positions in SEQ ID NO:2 as found in laboratory, various clades, and/or clinical isolates of HIV.
  • native sequence when used herein for purposes of the specification and claims and in reference to the amino acid sequence of the HR2 region of HIV gp41 , means a naturally occurring sequence found in laboratory HIV strains and/or HIV clinical isolates. Such sequences are readily available from public gene databases such as GenBank. For purposes of illustration, but not limitation, some of such native sequences are illustrated in FIG. 2, in which illustrative substitutions (e.g., polymorphisms) are noted in various amino acid positions in the amino acid sequence represented by SEQ ID NO:2.
  • base sequence when used herein for purposes of the specification and claims, refers to the native sequence (or a peptide consisting of the native sequence) from which is derived a synthetic peptide according to the present invention.
  • a synthetic peptide is derived from the base sequence in that the synthetic peptide comprises some of the amino acid sequence of the base sequence, in addition to differing from the base sequence by the addition of either (a) a plurality of helix-promoting amino acids into the amino acid sequence of the synthetic peptide, or (b) a combination of helix-promoting amino acids and charged amino acids (in forming ion pairs) into the amino acid sequence of the synthetic peptide; to impart the unexpected, improved biological activity of the synthetic peptide, as compared to the base sequence not containing such added amino acids.
  • reactive functionality when used herein for purposes of the specification and claims, means a chemical group or chemical moiety that is capable of forming a covalent bond and/or is protective (e.g., protects peptide derivatives from reacting with themselves or other molecules).
  • a reactive functionality is known to those skilled in the art to comprise a group that includes, but is not limited to, maleimide, thiol, carboxy, phosphoryl, acyl, hydroxyl, acetyl, hydrophobic, amido, dansyl, fluorenylmethyoxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), sulfo, a succinimide, a thiol-reactive, an amino-reactive, a carboxyl-reactive, and the like.
  • a chemical group, added to the N-terminal amino acid of a synthetic peptide to block chemical reactivity of that amino terminus of the peptide comprises an N- terminal group.
  • N-terminal groups for protecting the amino terminus of a peptide are well known in the art, and include, but are not limited to, lower alkanoyl groups, acyl groups, sulfonyl groups, and carbamate forming groups.
  • Preferred N-terminal groups may include acetyl, Fmoc, and Boc.
  • a chemical group, added to the C- terminal amino acid of a synthetic peptide to block chemical reactivity of that carboxy terminus of the peptide comprises a C-terminal group.
  • Such C-terminal groups for protecting the carboxy terminus of a peptide are well known in the art, and include, but are not limited to, an ester or amide group.
  • a chemical moiety may comprise a linker.
  • Linkers are known to refer to a compound or moiety that acts as a molecular bridge to operably link two different molecules (e.g., a wherein one portion of the linker binds to a peptide according to the present invention, and wherein another portion of the linker binds to a macromolecular carrier or another antiviral peptide known to inhibit HIV transmission to a target cell).
  • the two different molecules may be linked to the linker in a step-wise manner.
  • Linkers are known to those skilled in the art to include, but are not limited to, chemical chains, chemical compounds (e.g., reagents), and the like.
  • the linkers may include, but are not limited to, homobifunctional linkers and heterobifunctional linkers.
  • Heterobifunctional linkers well known to those skilled in the art, contain one end having a first reactive functionality to specifically link a first molecule, and an opposite end having a second reactive functionality to specifically link to a second molecule.
  • linker may be employed as a linker with respect to the present invention.
  • the linker may vary in length and composition for optimizing such properties as preservation of biological function stability, resistance to certain chemical and/or temperature parameters, and of sufficient stereo-selectivity or size.
  • the linker should not significantly interfere with the ability of the peptide according to the present invention (to which it is linked) to function as an inhibitor of either or both of HIV fusion and HIV transmission to a target cell.
  • a preferred reactive functionality may be used to the exclusion of reactive functionalities other than the preferred reactive functionality.
  • Such macromolecular carriers are well known in the art to include, but are not limited to, serum proteins, polymers, carbohydrates, and lipid-fatty acid conjugates.
  • Serum proteins typically used as macromolecular carriers include, but are not limited to, transferrin, albumin (preferably human), immunoglobulins (preferably human IgG or one or more chains thereof), or hormones.
  • Polymers typically used as macromolecular carriers include, but are not limited to, polylysines or poly(D-L- alanine)-poly(L-lysine)s, or polyols.
  • a preferred polyol comprises a water-soluble poly(alkylene oxide) polymer, and can have a linear or branched chain.
  • Suitable polyols include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), and PEG-PPG copolymers.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PEG-PPG copolymers PEG-PPG copolymers.
  • a preferred polyol comprises PEG having an average molecular size selected from the range of from about 1 ,000 Daltons to about 20,000 Daltons.
  • Other types of macromolecular carriers that can be used, which generally have molecular weights higher than 20,000, are known in the art.
  • synthetic in relation to a peptide according to the present invention, is used hereinafter for the purposes of the specification and claims to mean that the peptide is produced by chemical synthesis, recombinant expression, biochemical or enzymatic fragmentation of a larger molecule, chemical cleavage of larger molecule, a combination of the foregoing or, in general, made by any other method in the art, and isolated.
  • isolated when used in reference to a peptide, means that the synthetic peptide is substantially free of components which have not become part of the integral structure of the peptide itself; e.g., such as substantially free of cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized or produced using biochemical or chemical processes.
  • electrode pair when used herein for purposes of the specification and claims, is customarily referring to a simple electrostatic interaction between oppositely charged ions (e.g., between two oppositely charged amino acids) in an amino acid sequence.
  • Each oppositely charged ion is on a side chain of an amino acid.
  • a "salt bridge" is an ion pair in close spatial relationship (as known in the art), as determined by nuclear magnetic resonance or other standard method known in the art.
  • an ion pair is formed by 2 oppositely charged amino acid residues spaced apart by either three amino acids (i.e., in an / ' ,/ ' + 4 arrangement) or two amino acids (i.e., in an / ' ,/ ' + 3 arrangement) in a contiguous sequence contained in an amino acid sequence in forming a helix.
  • a positively charged amino acid e.g., lysine, arginine, histidine
  • a negatively charged amino acid e.g., glutamic acid, aspartic acid
  • a synthetic peptide is derived from a base sequence except that in the place of a neutral (charge) or negatively charged amino acid (as in the base sequence) included in a corresponding position in the synthetic peptide is a positively charged amino acid placed such that an ion pair is formed (e.g., in an /,/ + 3 arrangement or /,/ + 4 arrangement) with a negatively charged amino acid.
  • a synthetic peptide is derived from a base sequence except that in the place of a neutral (charge) or positively charged amino acid (as in the base sequence), included in the synthetic peptide is a negatively charged amino acid in a position in the amino acid sequence such that an ion pair is formed (e.g., in an / ' ,/ ' + 3 arrangement or /,/ ' + 4 arrangement) with a positively charged amino acid.
  • both a negatively charged amino acid and a positively charged amino acid are included in the synthetic peptide in an arrangement such that an ion pair is formed in the synthetic peptide, which ion pair is absent in the base sequence from which the synthetic peptide is derived.
  • improved biological activity when used herein for purposes of the specification and claims in reference to a synthetic peptide according to the present invention, means that (a) increased is the antiviral activity (e.g., as measured by the IC50 or other measurement standard in the art for measuring antiviral potency) of a synthetic peptide, as compared to the antiviral activity of a base sequence from which it was derived, against HIV strains showing reduced susceptibility ("resistance") to the antiviral activity of the base sequence; or (b) increased is the antiviral activity of a synthetic peptide, as compared to the antiviral activity of a base sequence from which the synthetic peptide is derived, against HIV strains showing resistance to the antiviral activity of the base sequence; and improved is the pharmacokinetic properties (e.g., as measured by one or more parameters such as Area Under the Curve (AUC), biological half-life, and or clearance; or other measurement standard in the art for measuring pharmacokinetic properties) of a synthetic peptide
  • AUC Area
  • improved biological activity comprises an increase in antiviral activity preferably no less than 20 fold the activity, as that observed for the base sequence and in relation to virus isolates (mutants) which are resistant to the base sequence.
  • improved biological activity comprises an IC50 of less than or equal to 0.500 ⁇ g/ml against virus isolates resistant to the base sequence. More preferably, such IC50 of the n synthetic peptide is in the nanogram/ml or picogram/ml range.
  • the base sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and a combination thereof.
  • a synthetic peptide has improved pharmacokinetic properties when the synthetic peptide has one or more of (a) a longer biological half life (t Yz), and (b) a reduction in biological clearance (Cl); as compared to that of a base sequence from which it is derived.
  • the synthetic peptide typically allows for a clearance that is reduced by no less than 30 percent relative to that of a base sequence from which it is derived, as will be shown in more detail in the examples herein.
  • the synthetic peptide typically allows for an increase in biological half-life of no less than 5 fold as compared to the biological half-life of a base sequence from which it is derived, as will be shown in more detail in the examples herein.
  • pharmacokinetic properties when used herein for purposes of the specification and claims, means the total amount of active ingredient (e.g., synthetic peptide analog) in a pharmaceutical composition that is systematically available over time. Pharmacokinetic properties may be determined by measuring total systemic concentrations of synthetic peptide analog over time after administration, either singularly or in comparison with pharmacokinetic properties after administration of synthetic peptide alone (i.e., with no amide-forming amino acid operably bound thereto).
  • pharmacokinetic properties may be expressed in terms of the Area Under the Curve (AUC), biological half-life, and/or clearance.
  • AUC is the integrated measure of systemic active ingredient concentrations over time, in units of mass x time/volume. Following the administration of a dose of active ingredient, the AUC from the time of dosing to the time when no active ingredient remains in the body, is a measure of the exposure of the individual to the active ingredient (and/or a metabolite of an active ingredient). Clearance is defined as dose/AUC, and is expressed in units of volume/weight/time.
  • stability when used herein for purposes of the specification and claims in reference to a synthetic peptide according to the present invention, means the stability of the alpha-helical coiled coil structure of the peptide. It is known by those skilled in the art that stability can be measured by standard methods known in the art, such as by determining the melting temperature ("Tm") of the peptide (see, e.g., Example 1 herein).
  • Tm melting temperature
  • a synthetic peptide comprising a plurality of amino acid substitutions as described herein, and as compared to a base sequence from which the synthetic peptide is derived, demonstrates greater stability as may be discerned by observing a higher melting temperature of the synthetic peptide, as compared to the melting temperature of the base sequence from which the synthetic peptide was derived.
  • Such one or more amino acid substitutions may include introduction of helix-promoting amino acids in a proper position (e.g., in replacing side chains of lower helix propensity); or introduction helix-promoting amino acids, and charged amino acids (in forming ion pairs), in one or more heptad repeats that serve to stabilize the coiled coil.
  • a Reference Sequence may be a synthetic peptide having an amino acid sequence of any one of SEQ ID NOs: 5-98
  • a Compared Sequence is a synthetic peptide which is compared to the Reference Sequence for percent identity.
  • treatment or “therapy”, are used interchangeably with respect to HIV infection, and for purposes of the specification and claims, to mean that a synthetic peptide (or a composition having the synthetic peptide as an active drug substance) may be used to affect one or more processes associated with HIV infection, or one or more parameters or endpoints used as indicators for determining the therapeutic effect of such treatment or therapy (e.g., "therapeutic application”).
  • the synthetic peptide may be used to inhibit one or more of the following processes: transmission of HIV to a target cell; fusion between HIV and a target cell ("HIV fusion"); viral entry (the process of HIV or its genetic material entering into a target cell during the infection process); and syncytia formation (e.g., between an HIV-infected cell, and a target cell).
  • HIV fusion the process of HIV or its genetic material entering into a target cell during the infection process
  • syncytia formation e.g., between an HIV-infected cell, and a target cell.
  • Viral suppression (determined by methods known in the art for measuring the viral load of HIV in a body fluid or tissue) is a commonly used primary endpoint, and an increase in the number of CD4 + cells circulating in the bloodstream is a commonly used secondary endpoint, for assessing the efficacy of a drug in treatment or therapy of HIV infection; each being a measurable effect of inhibiting transmission of HIV to a target cell.
  • a synthetic peptide may be used to effect a therapeutic application comprising viral suppression and/or an increase in the relative number of circulating CD4 + cells.
  • a synthetic peptide of the present invention comprises the following distinguishing and functional characteristics.
  • a synthetic peptide according to the present invention is derived from the native sequence of the HR2 region of HIV-1 gp41, and more particularly comprises any one or more of SEQ ID NO:s 2, 3, or 4 as a base sequence; however, the synthetic peptide differs from the base sequence by the inclusion in the amino acid sequence of the synthetic peptide of a plurality of amino acid substitutions (as compared to the corresponding positions in the base sequence from which the synthetic peptide is derived) which result in an unexpected, improved biological activity, and may further comprise an increase in helicity, as compared to such base sequence from which the synthetic peptide is derived.
  • the synthetic peptide differs from the base sequence by: (a) by substitution of between about 5% and about 50% of the amino acids of the base sequence with a helix-promoting amino acid (e.g., adding an amino acid having a greater helical propensity in place of an amino acid having a lower helical propensity than the amino acid replacing it, or adding an uncharged helix-promoting amino acid in place of a charged amino acid); and (b) comprising improved biological activity.
  • a helix-promoting amino acid e.g., adding an amino acid having a greater helical propensity in place of an amino acid having a lower helical propensity than the amino acid replacing it, or adding an uncharged helix-promoting amino acid in place of a charged amino acid
  • Such synthetic peptides are exemplified by a synthetic peptide having an amino acid sequence of SEQ ID NO:5, or an amino acid sequence having 90% identity with SEQ ID NO:5 and differing from the base sequence by (i) an addition of a plurality of helix-promoting amino acids as compared to corresponding amino acid positions in the base sequence from which the synthetic peptide is derived, and (ii) improved biological activity.
  • the synthetic peptide has two or more substitutions that include at least an "a" position in one heptad and a "d" position in a different (preferably adjacent) heptad surprisingly resulting in improved biological activity, as compared to the base sequence from which the synthetic peptide is derived.
  • the total number of "a" and “d” positions of the base sequence which are substituted with a helix- promoting amino acid ranges from 2 to 5.
  • the helix-promoting amino acid is either a leucine or isoleucine, or a combination of leucine and isoleucine, in forming from 1 to 3 additional leucine zipper-like motifs as compared to the base sequence from which the synthetic peptide is derived.
  • Such synthetic peptides are exemplified by SEQ ID NOs:82, 84, 85, 86, and 87.
  • the synthetic peptide differs from the base sequence by (a) substitution of between about 5% and about 60% of the amino acids of the base sequence with (i) a helix-promoting amino acid, and (ii) a charged amino acid residue, resulting in formation of a plurality of ion pairs in the synthetic peptide which were not present in the base sequence from which the synthetic peptide is derived, and more preferably in an arrangement wherein the synthetic peptide comprises a number of ion pairs ranging from about 3 ion pairs to about 10 ion pairs; and (b) comprising improved biological activity.
  • a synthetic peptide is exemplified by a synthetic peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37
  • a synthetic peptide according to the present invention is not any one of SEQ ID NOs:96-98.
  • a preferred synthetic ' peptide may be used to the exclusion of synthetic peptide other than the preferred synthetic peptide.
  • the synthetic peptide according to the present invention may further comprise one or more of: an additional 1 to about 20 amino acids added at the N-terminus of (e.g., at the N-terminal amino acid corresponding to) the base sequence from which the synthetic peptide is derived; a deletion of from about 1 to 10 amino acids from the N- terminus (e.g., N-terminal end and inward) of the base sequence from which the synthetic peptide is derived; an additional 1 to about 20 amino acids at the C-terminus of (e.g., at the C-terminal amino acid corresponding to) the base sequence from which the synthetic peptide is derived; and a deletion of from about 1 to 10 amino acids from the C- terminus (e.g.,
  • base sequences (SEQ ID NOs. 2, 3, and 4) share the following amino acid sequence (SEQ ID NO:3).
  • a synthetic peptide comprises, as compared to a base sequence from which it is derived, the addition of a plurality of helix-promoting amino acids in substituting for amino acids (e.g. of less helical propensity, or charged amino acids) present in the base sequence to result in a synthetic peptide having improved biological activity.
  • substitutions include the following, wherein an "h" under the amino acid position indicates addition of a helix-promoting amino acid in place of a charged amino acid (e.g., glutamic acid) or an amino acid of less helical propensity (including, but not limited to amino acids considered to have no helical propensity) in the corresponding amino acid position of SEQ ID NO:3.
  • a charged amino acid e.g., glutamic acid
  • amino acid of less helical propensity including, but not limited to amino acids considered to have no helical propensity
  • the synthetic peptide has two or more substitutions that include at least an "a" position in one heptad and a "d" position in a different (preferably adjacent) heptad surprisingly resulting in improved biological activity, as compared to the base sequence from which the synthetic peptide is derived. More preferably, the total number of "a” and "d" positions of the base sequence which are substituted with a helix- promoting amino acid ranges from 2 to 5.
  • the helix-promoting amino acid is either a leucine or isoleucine, or a combination of leucine and isoleucine, in forming from 1 to 3 additional leucine zipper-like motifs as compared to the base sequence from which the synthetic peptide is derived.
  • the synthetic peptides according to the present invention have a plurality of additional amino acids as compared to (e.g., substituted for amino acids in) the base sequence consisting of an amino acid sequence of SEQ ID NO:3, which include, but are not limited to, any one of more of the following; wherein a "c" under the amino acid position indicates addition of a charged amino acid in place of an uncharged amino acid in the corresponding amino acid position of SEQ ID NO:3 for forming an ion pair with an appropriately spaced apart oppositely charged amino acid (i.e., charge opposite to the amino acid added); and an "h" under the amino acid position indicates addition of a helix-promoting amino acid in place of a charged amino acid (e.g., glutamic acid) or an amino acid of less helical propensity (including, but not limited to amino acids considered to have no helical propensity) in the corresponding amino acid position of SEQ ID NO:3.
  • a "c" under the amino acid position indicates addition of a charged amino acid
  • Examples of ion pairs that may be formed in the amino acid positions following substitutions with charged (where "+” represents positively charged, and “-” represents negatively charged) amino acids (indicated by “c") include, but are riot limited to, any one or more of the following.
  • a synthetic peptide according to the present invention (when compared to a base sequence from which it is derived) has an amino acid sequence having: (a) no less than 2 helix-promoting amino acids, and no more than 14 helix-promoting amino acids, in positions of which corresponding positions of the base sequence lack a helix-promoting amino acid; and (b) no less than 2 charged amino acids, and no more than 10 charged amino acids, in positions of which corresponding positions of the base sequence lack a charged amino acid.
  • Helicity is a biophysical parameter.
  • the helicity of peptides consisting of a base sequence typically is in a range of from about 9% to about 10%, as assessed by circular dichroism (See Example 1 , herein).
  • Synthetic peptides according to the present invention generally have a helicity that is in a range of from about 25% to about 100%, and preferably in a range of from about 48% to about 85%.
  • a synthetic peptide according to the present invention may comprise a sequence of no less than about 15 amino acids and no more than about 60 amino acid residues in length, and preferably no less than 28 amino acids and no more than about 38 amino acids in length.
  • a synthetic peptide according to the present invention is derived from (e.g., comprises a contiguous sequence of at least the contiguous amino acid residues) any one or more of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, or a portion thereof, with inclusion in the synthetic peptide of some amino acids which are different from (substitutions of amino acids in) amino acids in the corresponding position of the base sequence from which the synthetic peptide is derived.
  • the differences in the amino acid sequence have been found to influence biophysical (e.g., helicity and stability) and biological (e.g., antiviral) parameters described herein in more detail.
  • the synthetic may further comprise one or more conservative substitutions, as compared to the base sequence from which it is derived.
  • a synthetic peptide according to the present invention may further comprise a macromolecular carrier.
  • a single mutation particularly in a codon for the connecting loop of the HIV-1 reverse transcriptase fingers subdomain (e.g., at codon 69 or codon 151 ) is associated with broad cross resistance to all nucleoside reverse transcriptase inhibitors.
  • NRTI non-nucleoside reverse transcriptase inhibitors
  • a single mutation at amino acid 103 which is believed to be in the hydrophobic cavity or NNRTI binding site of the reverse transcriptase results in broad cross-resistance to NNRTIs.
  • protease inhibitors Pro
  • HIV-1 strains have emerged possessing cross- resistance to all members of this class.
  • synthetic peptides derived from the HR2 region and which have been modified in the amino acid sequence according to the present invention, can demonstrate improved biological activity (i.e., increased antiviral potency) against viral mutations which render the virus resistant to peptides derived from the native sequence of HIV gp41.
  • a synthetic peptide according to the present invention comprises unexpected, improved biological activity when, against a virus resistant to peptides derived from the native sequence of HIV-1 gp41 (e.g., a base sequence, or T20 (SEQ ID NO:1 )), it demonstrates an IC50 of less than or equal to 0.3 ⁇ g/ml, and preferably, less than 0.10 ⁇ g/ml.
  • a synthetic peptide according to the present invention does not consist of any one of SEQ ID NOs: 96, 97, or 98. Additionally, in a preferred embodiment, a synthetic peptide according to the present invention has both an increase in antiviral activity against a virus resistant to peptides derived from the native sequence of HIV-1 gp41 (e.g., a base sequence from which the synthetic peptide is derived), and improved pharmacokinetic properties as compared to a base sequence from which the synthetic peptide is derived.
  • the clearance values are each greater than 0.30, as measured using the methods described herein.
  • a synthetic peptide according to the present invention may have a clearance value that ranges from about 0.005 to about 0.07 (expressed in L/K/hr).
  • the improved pharmacokinetic properties are illustrated by no less than a 30% reduction in clearance.
  • the biological half-life (also termed herein as "terminal elimination half-life” or "t Yz"; expressed in hours (hr) or fraction thereof) are each less than 0.50 hr, as measured using the methods described herein.
  • a synthetic peptide according to the present invention may have a biological half-life that ranges from about 3 hr to greater than 20 hr.
  • a synthetic peptide according to the present invention comprises a stability represented by a melting temperature (“Tm") in the range of from about 25°C to about 75°C, and more preferably from about 36°C to about 65°C.
  • Tm melting temperature
  • a synthetic peptide may further comprise a component selected from the group consisting of one or more reactive functionalities (e.g., at either the C-terminal end, or N-terminal end, or a combination thereof (both the C-terminal end and N-terminal end)), a pharmaceutically acceptable carrier, a macromolecular carrier, and a combination thereof.
  • reactive functionalities e.g., at either the C-terminal end, or N-terminal end, or a combination thereof (both the C-terminal end and N-terminal end)
  • a pharmaceutically acceptable carrier e.g., at either the C-terminal end, or N-terminal end, or a combination thereof (both the C-terminal end and N-terminal end)
  • a pharmaceutically acceptable carrier e.g., at either the C-terminal end, or N-terminal end, or a combination thereof (both the C-terminal end and N-terminal end)
  • macromolecular carrier e.g., a combination thereof
  • Peptides including synthetic peptides and base sequences, were synthesized on a peptide synthesizer using standard solid-phase synthesis techniques and using standard FMOC peptide chemistry.
  • the synthetic peptides may further comprise reactive functionalities; i.e., most were blocked at the N-terminus by an acetyl group and/or at the C-terminus by an amide group, or comprised a linker at the N- terminus or C terminus.
  • the peptides were precipitated, and the precipitate was lyophilized.
  • the peptides were then purified using reverse-phase high performance liquid chromatography; and peptide identity was confirmed with electrospray mass spectrometry.
  • CD circular dichroism
  • Raw ellipticity values were converted to mean residue ellipticity using standard methods, and plotted was the wavelength (from 200 to 260 nm) versus [ ⁇ ] x 10-3 (degrees cm 2 /dmol). Percent helicity values were then calculated using standard methods (usually expressed as percent helicity at 10 ⁇ M, 25°C). Assessment of thermal stability was performed by monitoring the change in CD signal at 222 nm as temperature was raised in 2°C steps, with 1 minute equilibration times. The stability for each sample (e.g., synthetic peptide), as represented by the Tm value, is the temperature corresponding to the maximum value of the first derivative of the thermal transition.
  • antiviral activity e.g., one measure being the ability to inhibit transmission of HIV to a target cell
  • an in vitro assay which has been shown, by data generated using peptides derived from the HR regions of HIV gp41 , to be predictive of antiviral activity observed in vivo. More particularly, antiviral activity observed using an in vitro infectivity assay ("Magi-CCR5 infectivity assay"; see, e.g., U.S. Patent No.
  • the number of stained nuclei can thus be interpreted as equal to the number of infectious virions in the challenge inoculum if there is only one round of infection prior to staining.
  • Infected cells are enumerated using a CCD-imager and both primary and laboratory adapted isolates show a linear relationship between virus input and the number of infected cells visualized by the imager.
  • IC50 is defined as the concentration of active ingredient resulting in a 50% reduction in infectious virus titer).
  • Peptides tested for antiviral activity were diluted into various concentrations, and tested in duplicate or triplicate against an HIV inoculum adjusted to yield approximately 1500-2000 infected cells/well of a 48 well microtiter plate.
  • the peptide in the respective dilution was added to the cMAGI or MAGI cells, followed by the virus inocula; and 24 hours later, an inhibitor of infection and cell- cell fusion (e.g., T20) was added to prevent secondary rounds of HIV infection and cell- cell virus spread.
  • the cells were cultured for 2 more days, and then fixed and stained with the X-gal substrate to detect HIV-infected cells.
  • Viruses resistant to the antiviral activity of a peptide consisting of a base sequence can be produced using standard laboratory methods. Basically, after calculating the IC50 and IC90, cells were mixed with virus and the peptide (e.g., at a concentration close to the IC90) in culture (including when the cells are split thereafter). The cultures are maintained and monitored until syncytia are present.
  • Virus harvested from this first round of culture is used to infect cells in a second round of culture, with the peptide present in a higher concentration (2 to 4 times) than that used in the first round of culture.
  • the second round of culture is maintained and monitored for presence of virus resistant to the antiviral activity of the peptide.
  • Subsequent rounds of culture may be needed to finally generate a viral isolate resistant to the antiviral activity of the peptide (at a pre-determined level of the IC50 of the peptide against such isolate).
  • a synthetic peptide or a base sequence from which a synthetic peptide is derived was dosed intravenously in cynomolgus monkeys (Macaca fasicularis) (other animal models may be used for determining pharmacokinetic properties, as known in the art).
  • cynomolgus monkeys Macaca fasicularis
  • other animal models may be used for determining pharmacokinetic properties, as known in the art.
  • blood samples were drawn and plasma isolated by centrifugation. Plasma samples were stored frozen until analysis by LC-MS (liquid chromatography/mass spectrometry) in the electrospray, positive-ion mode.
  • a synthetic peptide or base sequence was eluted from a C18 HPLC column with a gradient of acetonitrile in a buffer of 10 mM ammonium acetate, pH 6.8.
  • plasma samples were deproteinated with either two or three volumes of acetonitrile containing 0.5 % formic acid.
  • Duplicate calibration standards in cynomolgus plasma samples were prepared at the same time as the samples and analyzed before and after the samples containing either synthetic peptide or base sequence.
  • Pharmacokinetic properties were calculated from the plasma concentration-time data using either mono-exponential or bi-exponential mathematical models. Models were derived by non-linear least squares optimization. A 1/C 2 weighting of concentrations was used. The following equations were used to calculate area-under the plasma concentration vs. time curve (AUC), total body clearance (Cl), and terminal elimination half-life (t V .
  • the synthetic peptide according to the present invention was synthesized to comprise a plurality of helix-promoting amino acid substitutions in relation to base sequence consisting of SEQ ID NO:4.
  • synthetic peptide according to the present invention was compared to peptides having a base sequence of either SEQ ID NO:2 or SEQ ID NO:4 for biophysical parameters and biological parameters, as determined using the methodology described in Example 1 herein.
  • virus mutants which are resistant to the antiviral activity of peptides having the base amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 (the resistant viral isolate being designated as "RY" in Table 1 and subsequent Tables).
  • a synthetic peptide according to the present invention (a) demonstrates an increase in helicity (e.g., an increase in a range of from about 3 fold to about 5 fold or greater); and (b) demonstrates a significant increase in antiviral activity, an unexpected, improved biological activity, against virus resistant to peptides having any one of the base sequences (e.g., SEQ ID NOs:2 or 4) (e.g., virus isolate HIV RY).
  • EXAMPLE 3 In another embodiment, produced was a synthetic peptide comprising an addition of amino acids comprising helix-promoting amino acids and charged amino acids (in forming a plurality of ion pairs), in place of amino acids present in any one or more of base sequences SEQ ID NOs: 2-4.
  • synthetic peptides exemplified by an amino acid sequence having any one of SEQ ID NOs:6-81 , and 83-95 were produced and assessed using the methods outlined in Example 1 herein.
  • a synthetic peptide according to the present invention demonstrates a significant increase in antiviral activity, an unexpected, improved biological activity, against virus resistant to peptides having any one of the base sequences (e.g., SEQ ID NOs:2 or 4) (e.g., virus isolate HIV RY). Additionally, a synthetic peptide may further demonstrates an increase in helicity (e.g., in a range of from about 3 fold to about 5 fold or greater), as compared to any one of the base sequences (e.g., SEQ ID NOs:2 or 4).
  • a synthetic peptide according to the present invention additionally (e.g., in addition to demonstrating an increase in helicity and unexpected, improved biological activity) and preferentially demonstrates a stability as, for example, measured by a Tm in the range of from about 25°C to about 75°C, and more preferably from about 36°C to about 65°C.
  • Tables 1 and 2 demonstrate the unexpected, improved biological activity of a synthetic peptide according to the present invention, as assessed by (a) determining the antiviral activity of the synthetic peptide against an HIV strain that demonstrates resistance to the activity of a base sequence from which the synthetic peptide is derived; and (b) demonstrating the synthetic peptide has antiviral activity, as measured by an IC50 of less than 0.10 ⁇ g/ml, against the HIV strain that demonstrates resistance to the activity of a base sequence from which the synthetic peptide is derived.
  • synthetic peptide according to the present invention was used in attempts to generate resistant virus in vitro.
  • an in vitro culture of HIV-infected cells was passaged in the presence of either the individual synthetic peptide or the base sequence in efforts to reach an endpoint of generation of an isolate of HIV which was resistant at a concentration of 10 ⁇ g/ml to 20 ⁇ g/ml of the peptide with which the HIV-infected cells were incubated.
  • the synthetic peptide or base sequence typically, starting with the synthetic peptide or base sequence at a concentration between it's IC50 and IC90, the HIV-infected cells were cultured in vitro, and split every 2 to 3 days adding the synthetic peptide or base sequence during the split to maintain the HIV-infected cells in the presence of a constant and consistent amount of synthetic peptide or base sequence.
  • a resistant isolate as measured by cytopathic effect/syncytia formation; considered as one passage
  • cells were infected with the resistant isolate generated from that passage, and the cells were then cultured in the presence of a higher concentration (e.g., 2. to 3 times the concentration used in the previous passage) of synthetic peptide or base sequence until a resistant HIV isolate was generated. This procedure was repeated until achieved is the endpoint. Determined is the number of passages, and number of days (number of days in each successful passage (where a viable virus was generated) then totaled together for all successful passages), in culture required to reach the endpoint. Results were averaged for each base sequence or synthetic peptide illustrated.
  • NA endpoint not achieved
  • EXAMPLE 4 Illustrated in this example is the improved pharmacokinetic properties of a synthetic peptide according to the present invention as compared to a base sequence from which the synthetic peptide is derived.
  • Table 4 illustrates pharmacokinetic properties of a representation of synthetic peptides as compared to the pharmacokinetic properties of a base sequence consisting of either SEQ ID NO:2 or SEQ ID NO:3.
  • a synthetic peptide as compared to a base sequence from which it was derived, exhibited a marked improvement in pharmacokinetic properties as observed in one or more pharmacokinetic properties (e.g., in either or both of clearance and V/).
  • the improved pharmacokinetic properties are illustrated by no less than a 30% reduction in clearance.
  • the improved pharmacokinetic properties of a synthetic peptide are illustrated by no less than a 5 fold increase in biological half-life; preferably, no less than a 10 fold increase in biological half-life; and more preferably, no less than a 30 fold increase in biological half-life.
  • EXAMPLE 5 The present invention provides for synthetic peptides according to the present invention, which possess antiviral activity as evidenced by their ability to inhibit transmission of HIV (including, unexpectedly, isolates resistant to a base sequence from which synthetic peptide was derived) to a target cell (e.g., see Tables 1 & 2). Additionally, provided are uses of synthetic peptide according to the present invention. For example, a synthetic peptide according to the present invention may be used as an active therapeutic substance in therapy of HIV infection. Also, a synthetic peptide according to the present invention may be used for the manufacture of a medicament for a therapeutic application comprising treatment of HIV.
  • a synthetic peptide according to the present invention may be used for treatment of HIV, including a therapeutic application thereof (e.g., reducing the viral load of HIV, and/or increasing the CD4+ cell population, in a treated individual).
  • a method of treating an HIV-infected individual comprises administering to the individual an amount of a synthetic peptide (including a composition/medicament in which the synthetic peptide is an active therapeutic substance) effective to treat the individual or to achieve the desired therapeutic application.
  • a baseline value from measuring the parameter of viral load and/or CD4+ cell count) is obtained from a clinical sample prior to treatment with the synthetic peptide.
  • test value One or more clinical samples are obtained subsequent to the initiation of treatment with synthetic peptide, and from such sample(s) is measured the parameter ("test value").
  • the baseline value and test value are compared to determine if the desired therapeutic application was achieved from treatment with synthetic peptide (e.g., a difference between the test value and a baseline value may be an indication that the desired therapeutic application was achieved).
  • a method for inhibiting transmission of HIV to a target cell comprising adding to the virus and the cell an amount of synthetic peptide according to the present invention effective to inhibit infection of the cell by HIV.
  • a method for inhibition of transmission of HIV to a cell comprising contacting the virus in the presence of a cell with an amount of synthetic peptide according to the present invention effective to inhibit infection of the cell by HIV.
  • a method for inhibiting HIV fusion e.g., a process by which HIV gp41 mediates fusion between the viral membrane and cell membrane during infection by HIV of a target cell
  • HIV fusion comprising contacting the virus in the presence of a cell with an amount of synthetic peptide according to the present invention effective to inhibit HIV fusion.
  • an effective amount would be a dose sufficient (by itself and/or in conjunction with a regimen of doses) to reduce HIV viral load in the individual being treated.
  • HIV viral load there are several standard methods for measuring HIV viral load which include, but are not limited to, by quantitative cultures of peripheral blood mononuclear cells, by plasma HIV RNA measurements, and by measuring the viral nucleic acids by a quantitative method involving nucleic acid amplification using standard methods known in the art. Methods for determining CD4 + cell levels (a "CD4 + cell count”) are standard in the art.
  • Such methods include, but are not limited to, flow cytometry, immunoassay, magnetic separation followed by cell counting, immunocytochemical, and immunostaining.
  • Standards for HIV viral load and CD4 + cell counts which are indicative of various stages of HIV infection and AIDS are well known in the art.
  • One source for such standards is the Centers for Disease Control.
  • the synthetic peptides of the invention can be administered in a single administration, intermittently, periodically, or continuously, as can be determined by a medical practitioner using methods such as monitoring viral load and/or blood levels of synthetic peptide.
  • the synthetic peptides according to the present invention may be administered once or multiple times daily, or periodically during a week period, or periodically during a month period.
  • the synthetic peptides according to the present invention may show synergistic results or added therapeutic benefit of inhibiting transmission of HIV to a target cell, when used as a component in a combination or a therapeutic regimen (e.g., when used simultaneously, or in a cycling on with one drug and cycling off with another) containing one or more additional antiviral drugs used for treatment of HIV including, but not limited to, HIV entry inhibitors (e.g., other HIV fusion inhibitors (T20, T1249, and the like), CCR5 inhibitors, retrocyclins, etc.), HIV integrase inhibitors, reverse transcriptase inhibitors (e.g., nucleoside or nonnucleoside), protease inhibitors, viral-specific transcription inhibitors, viral processing inhibitors, HIV maturation inhibitors, inhibitors of uridine phosphorylating enzyme, HIV vaccines, and the like, as well known in the art.
  • HIV entry inhibitors e.g., other HIV fusion inhibitors (T20, T1249, and the like),
  • combinations of antiviral agents may be used which include one or more synthetic peptides according to the present invention, thus increasing the efficacy of the therapy, and lessening the ability of the virus to become resistant to the antiviral drugs.
  • Combinations may be prepared from effective amounts of antiviral agents (useful in treating of HIV infection) currently approved or approved in the future, which include, but are not limited to, abacavir, AZT, delaviridine, ddC, ddl, efavirenz, FTC, GS 840, HBY097, 3TC, nevirapine, d4T, FLT, emtricitabine, amprenivir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, PNU-140690, ritonavir, saquinavir, telinavir, tenofovir, adefovir, atazanavir, lopina
  • Effective dosages of these illustrative antiviral agents which may be used in combinations with synthetic peptide according to the present invention, are known in the art. Such combinations may include a number of antiviral agents that can be administered by one or more routes, sequentially or simultaneously, depending on the route of administration and desired pharmacological effect, as is apparent to one skilled in the art.
  • Effective dosages of the synthetic peptides of the invention to be administered may be determined through procedures well known to those in the art; e.g., by determining potency, biological half-life, bioavailability, and toxicity.
  • an effective synthetic peptide dosage range is determined by one skilled in the art using data from routine in vitro and in vivo studies well know to those skilled in the art.
  • in vitro infectivity assays of antiviral activity enables one skilled in the art to determine the mean inhibitory concentration (IC) of the synthetic peptide necessary to block some amount of viral infectivity (e.g., 50% inhibition, IC 50 ; or 90% inhibition, IC 90 ).
  • IC mean inhibitory concentration
  • Appropriate doses can then be selected by one skilled in the art using pharmacokinetic data from one or more standard animal models, so that a minimum plasma concentration (C[minj) of the peptide is obtained which is equal to or exceeds a predetermined IC value.
  • an exemplary dosage range of the synthetic peptide according to the present invention may range from no less than 0.1 ⁇ g/kg body weight and no more than 10 mg/kg body weight; preferably a dosage range of from about 0.1-100 ⁇ g/kg body weight; and more preferably, a dosage of between from about 10 mg to about 250 mg of synthetic peptide.
  • synthetic peptide according to the present invention further comprises a macromolecular carrier that causes synthetic peptide to remain active in the blood longer than synthetic peptide alone (i.e., in achieving a longer circulating plasma concentration)
  • the amount of synthetic peptide in the dosage may be reduced as compared to the amount of synthetic peptide in a formulation not containing macromolecular carrier, and/or administered less frequently than a formulation not containing macromolecular carrier.
  • compositions including a medicament, of the present invention (e.g., synthetic peptide, preferably with one or more of a pharmaceutically acceptable carrier and a macromolecular carrier) may be administered to an individual by any means that enables the active agent to reach the target cells (cells that can be infected by HIV).
  • the compositions of this invention may be administered by any suitable technique, including oral, parenteral (e.g., intramuscular, intra peritonea I, intravenous, or subcutaneous injection or infusion, intradermal, or implant), nasal, pulmonary, vaginal, rectal, sublingual, or topical routes of administration, and can be formulated in dosage forms appropriate for each route of administration.
  • the specific route of administration will depend, e.g., on the medical history of the individual, including any perceived or anticipated side effects from such administration, and the formulation of synthetic peptide being administered (e.g., the nature of the pharmaceutically acceptable carrier and/or macromolecular carrier of which synthetic peptide may further comprise).
  • the administration is by injection (using, e.g., intravenous or subcutaneous means), but could also be by continuous infusion (using, e.g., slow-release devices or minipumps such as osmotic pumps, and the like).
  • a formulation may comprise synthetic peptide according to the present invention which further comprises one or more of a pharmaceutically acceptable carrier and a macromolecular carrier; and may further depend on the site of delivery, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • a preferable formulation is one in which synthetic peptide according to the present invention is combined with or further comprises one or more of an agent, drug, reactive functionality, macromolecular carrier, or pharmaceutically acceptable carrier that inhibits or delays or retards the metabolism/degradation of synthetic peptide, particularly after it is administered to an individual.
  • a formulation may comprise nucleotide sequences encoding synthetic peptide according to the present invention, as described herein in more detail, which upon administration, is expressed in cells of interest using techniques and expression vectors well known in the art.
  • EXAMPLE 6 It is apparent to one skilled in the art, that based on the respective amino acid sequences of the synthetic peptides according to the present invention, that polynucleotides encoding such synthetic peptides may be synthesized or constructed, and that such synthetic peptides may be produced by recombinant DNA technology as a means of manufacture and/or (for example, in vivo production by introducing such polynucleotides in vivo as a means of gene or cell therapy) for a method of inhibiting transmission of HIV to a target cell.
  • polynucleotide sequence can encode a synthetic peptide according to the present invention, and that such polynucleotides may be synthesized on the basis of triplet codons known to encode the amino acids of the amino acid sequence of the synthetic peptide, third base degeneracy, and selection of triplet codon usage preferred by the host cell (e.g., prokaryotic or eukaryotic, species, etc,) in which expression is desired,
  • the host cell e.g., prokaryotic or eukaryotic, species, etc,
  • SEQ ID NO:99 is a polynucleotide encoding SEQ ID NO:2, a base sequence, from which, as apparent to one skilled in the art, codon usage will generally apply to polynucleotides encoding synthetic peptides of the present invention.
  • a polynucleotide encoding a synthetic peptide according to the present invention comprises a nucleic acid sequence encoding a synthetic peptide selected from the group consisting of SEQ ID NOs:5-98, or an amino acid sequence having at least (e.g., no less than) 90% identity with any one or more of SEQ ID NOs:5-98 and differing from a base sequence by (i) an addition of a plurality of helix-promoting amino acids as compared to corresponding amino acid positions in the base sequence from which the synthetic peptide is derived; or an addition of a plurality of helix-promoting amino acids as compared to corresponding amino acid positions in the base sequence from which the synthetic peptide is derived, and an addition of a plurality of charged amino acids as compared to the positions corresponding to the base sequence
  • a prokaryotic expression vector containing a polynucleotide encoding a synthetic peptide according to the present invention, and its use for the recombinant production of synthetic peptide.
  • the polynucleotide may be positioned in a prokaryotic expression vector so that when synthetic peptide is produced in bacterial host cells, it is produced as a fusion protein with sequences which assist in purification of the synthetic peptide.
  • Inclusion bodies may be separated from other prokaryotic cellular components by methods known in the art to include denaturing agents, and fractionation (e.g., centrifugation, column chromatography, and the like).
  • a nucleic acid sequence encoding a synthetic peptide according to the present invention can be inserted into a plasmid or vectors other than plasmids, and other expression systems can be used including, but not limited to, bacteria transformed with a bacteriophage vector, or cosmid DNA; yeast containing yeast vectors; fungi containing fungal vectors; insect cell lines infected with virus (e. g. baculovirus); and mammalian cell lines having introduced therein (e.g., transfected with) plasmid or viral expression vectors, or infected with recombinant virus (e.g.
  • the synthetic peptide requires that either the recombinant DNA molecule comprising the encoding sequence of the synthetic peptide, or the vector itself, contain the necessary control elements for transcription and translation which is compatible with, and recognized by the particular host system used for expression.
  • various promoters and enhancers can be incorporated into the vector or the recombinant DNA molecule comprising the encoding sequence to increase the expression of the synthetic peptide, provided that the increased expression of the synthetic peptide is compatible with (for example, non-toxic to) the particular host cell system used.
  • the selection of the promoter will depend on the expression system used. Promoters vary in strength, i.e., ability to facilitate transcription. Generally, for the purpose of expressing a cloned gene, it is desirable to use a strong promoter in order to obtain a high level of transcription of the gene and expression into gene product. For example, bacterial, phage, or plasmid promoters known in the art from which a high level of transcription has been observed in a host cell system comprising E.
  • coli include the lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P.sub.R and P.sub.L promoters, lacUV ⁇ , ompF, bla, Ipp, and the like, may be used to provide transcription of the inserted nucleotide sequence encoding the synthetic peptide.
  • mammalian promoters in expression vectors for mammalian expression systems are the promoters from mammalian viral genes. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • the host cell strain/line and expression vectors may be chosen such that the action of the promoter is inhibited until specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA (e.g., the lac operon is induced by the addition of lactose or isopropylthio-beta-D-galactoside ("IPTG"); trp operon is induced when tryptophan is absent in the growth media; and tetracycline can be use in mammalian expression vectors having a tet sensitive promoter).
  • expression of the synthetic peptide may be controlled by culturing transformed or transfected cells under conditions such that the promoter controlling the expression from the encoding sequence is not induced, and when the cells reach a suitable density in the growth medium, the promoter can be induced for expression from the encoding sequence.
  • Other control elements for efficient gene transcription or message translation are well known in the art to include enhancers, transcription or translation initiation signals, transcription termination and polyadenylation sequences, and the like.
  • synthetic peptide according to the present invention further comprises a macromolecular carrier.
  • macromolecular carriers are well known in the art to include, but are not limited to, serum proteins (the whole protein, or a substantial portion thereof), polymers, carbohydrates, and lipid-fatty acid conjugates, fatty acids, and the like.
  • Serum proteins typically used as macromolecular carriers include, but are not limited to, transferrin, albumin, immunoglobulins (preferably IgG or one or more chains thereof), or hormones; wherein the protein is preferably human, and more preferably a recombinant human protein.
  • Polymers typically used as macromolecular carriers include, but are not limited to, polylysines or poly(D-L-alanine)- poly(L-lysine)s, or polyols.
  • a preferred polyol comprises a water-soluble poly(alkylene oxide) polymer, and can have a linear or branched chain.
  • Suitable polyols include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), and PEG-PPG copolymers.
  • the macromolecular carrier may be conjugated to synthetic peptide.
  • the polyol in using a polyol, typically the polyol is derivatized or reacted with a coupling agent to form an "activated" polyol having one or more terminal reactive groups which can be used to react with a reactive functionality (e.g., preferably, a free amine group) of the synthetic peptide using methods standard in the art.
  • a reactive functionality e.g., preferably, a free amine group
  • Such reactive groups may include, but are not limited to, a hydroxy group, amino group, aldehyde group, and the like.
  • the polyol used may comprise a linear chain or branched chain polymer.
  • a synthetic peptide according to the present invention is synthesized, the last step of the synthesis process being the addition of a maleimide group (e.g., by a step in the solid phase synthesis of adding 3-maleimidoproprionic acid, washing, and then cleaving the synthetic peptide containing the maleimide group from the resin).
  • a maleimide group e.g., by a step in the solid phase synthesis of adding 3-maleimidoproprionic acid, washing, and then cleaving the synthetic peptide containing the maleimide group from the resin.
  • the synthetic peptide may then be administered (preferably, parenterally) to an individual such that the synthetic peptide conjugates to a macromolecular carrier such as a blood component (preferably, a serum protein, and more preferably, albumin).
  • a blood component preferably, a serum protein, and more preferably, albumin
  • recombinant human protein e.g., albumin, transferrin, immunoglobulin, or the like
  • recombinant human protein may be charged (“cationized") and then thiolated using standard coupling agents known in the art (e.g., using N-succinimidyl S-acetylthio-acetate).
  • the thiolated, charged recombinant human protein may be coupled to avidin using standard coupling reagents known in the art (using m-maleimidobenzoyl-N-hydroxysuccinimide ester).
  • the resultant avidinylated human protein may then be reacted with synthetic peptide which had been previously biotinylated using methods standard in the art.
  • the result is synthetic peptide that has been linked to macromolecular carrier.
  • the macromolecular carrier may be genetically expressed with synthetic peptide; e.g., as part of a fusion protein.
  • a DNA sequence encoding albumin may be cloned into a vector along with the DNA sequence encoding a linker and the DNA sequence encoding synthetic peptide according to the present invention, such that the resultant gene product is an albumin fusion protein comprising albumin with synthetic peptide linked at the C-terminal end, N-terminal end, or both the C-terminal and N-terminal ends of albumin.
  • yeast expression preferably for yeast expression
  • Useful yeast plasmid vectors are generally commercially available (e.g., pRS403-406 series and pRS413-416 series) and which may incorporate the yeast selectable markers (e.g., his3, trpl , Ieu2, ura3, and the like).
  • An expression vector containing a polynucleotide encoding an albumin-synthetic peptide fusion protein for yeast expression, may comprise an expression cassette comprising: a yeast promoter (e.g., a Sacchromyces PRB1 promoter); a sequence encoding a secretion leader which will facilitate secretion of the expressed gene product (e.g., could be the natural human albumin secretion leader and/or a yeast-derived secretion leader); a sequence encoding human albumin (e.g., as disclosed in Genbank); a sequence encoding a linker (e.g., the linker comprising a stretch of 5-20 amino acids, and more preferably amino acids that include glycine and serine); a polynucleotide encoding synthetic peptide; and a transcription terminator (e.g., Saccharomyces ADH1 ).
  • a yeast promoter e.g., a Sacchromyces PRB1 promoter
  • the polynucleotide encoding synthetic peptide would be placed between the promoter and the DNA encoding human albumin.
  • the resultant expression vector may then be used to transform yeast, and culture conditions for recombinant production, as well as purification of the recombinant product, could be performed using methods known in the art.
  • obtained can be synthetic peptide further comprising a macromolecular carrier.
  • a fusion protein containing a synthetic peptide according to the present invention was produced.
  • An expression vector was constructed which contained a polynucleotide (SEQ ID NO:102) encoding a fusion protein (SEQ ID NO: 103) comprising a maltose binding protein ("MBP"), a cleavable linker, and a synthetic peptide (SEQ ID NO:11 ) using standard methods known in the art.
  • the resultant expression vector was transformed into an E. coli strain as the host expression system, and the transformed cells were grown and then induced to express the fusion protein by the addition of IPTG to the bacterial culture using standard methods known in the art.
  • the induced bacterial cells were lysed with a microfluidizer, and the lysates were cleared of bacterial debris by centrifugation using standard methods known in the art.
  • the clarified lysate was then subjected to chromatography using columns packed with amylose resin for binding to the fusion protein (via the MBP portion). The columns were then washed, followed by elution of the fusion protein with a solution containing maltose.
  • the isolated fusion protein containing synthetic peptide tested for antiviral activity using the methods outlined in Example 1 , exhibited antiviral activity (e.g., an IC50 against HIV IIIB of ⁇ 0.10 ⁇ g/ml) against HIV-1.

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Abstract

L'invention concerne des peptides synthétiques basés sur une séquence native de la région HR2 de la gp41 du VIH. Ces peptides synthétiques présentent une pluralité de substitutions d'acides aminés comprenant (a) un acide aminé formant une hélice, ou (b) une combinaison d'acides aminés formant des hélices, et des acides aminés chargés introduits dans le peptide synthétique pour former des paires d'ions. Lesdits peptides synthétiques présentent une activité biologique améliorée inattendue, par rapport à un peptide avec une séquence d'acides aminés sans la pluralité de substitutions d'acides aminés. L'invention concerne également des polynucléotides codant pour les peptides synthétiques, et des méthodes d'utilisation desdits peptides synthétiques dans l'inhibition de la transmission du VIH dans une cellule cible, ou comme compositions destinées à inhiber la transmission du VIH dans une cellule cible.
PCT/US2004/042918 2004-01-07 2004-12-21 Peptides synthetiques derives de la hr2 de la gp41 du vih, et leur utilisation dans une therapie destinee a inhiber la transmission du virus de l'immunodeficience humaine WO2005067960A1 (fr)

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EP04815041A EP1708734A4 (fr) 2004-01-07 2004-12-21 Peptides synthetiques derives de la hr2 de la gp41 du vih, et leur utilisation dans une therapie destinee a inhiber la transmission du virus de l'immunodeficience humaine
AU2004313242A AU2004313242A1 (en) 2004-01-07 2004-12-21 HIV gp41 HR2-derived synthetic peptides, and their use in therapy to inhibit transmission of human immunodeficiency virus
BRPI0418304-5A BRPI0418304A (pt) 2004-01-07 2004-12-21 peptìdeo sintético, uso do mesmo, composição farmacêutica, seqüência de nucleotìdeos, e, métodos para inibição da transmissão de hiv para uma célula, para inibição da fusão de hiv, e para tratamento de um indivìduo infectado com hiv
JP2006549305A JP2007517522A (ja) 2004-01-07 2004-12-21 ＨＩＶｇｐ４１ＨＲ２由来の合成ペプチド、およびヒト免疫不全ウイルスの伝播を阻止する治療におけるその使用
CA002551082A CA2551082A1 (fr) 2004-01-07 2004-12-21 Peptides synthetiques derives de la hr2 de la gp41 du vih, et leur utilisation dans une therapie destinee a inhiber la transmission du virus de l'immunodeficience humaine
US11/477,574 US20060247416A1 (en) 2004-01-07 2006-06-29 HIV gp41 HR2-derived synthetic peptides, and their use in therapy to inhibit transmission of human immunodeficiency virus
IL176721A IL176721A0 (en) 2004-01-07 2006-07-05 HIV gp41 HR2-DERIVED SYNTHETIC PEPTIDES, AND THEIR USE IN THERAPY TO INHIBIT TRANSMISSION OF HUMAN IMMUNODEFICIENCY VIRUS
NO20063556A NO20063556L (no) 2004-01-07 2006-08-04 HIV gp41 HR2-avledede syntetiske peptider og deres anvendelse ved behandling av human mangel pa immunitet mot virus

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JP2009512655A (ja) * 2005-10-21 2009-03-26 エフ．ホフマン−ラ ロシュ アーゲー ペプチド−免疫グロブリン−複合体
WO2007045463A1 (fr) * 2005-10-21 2007-04-26 F.Hoffmann-La Roche Ag Conjugue peptide-immunoglobuline
WO2007068163A1 (fr) * 2005-12-14 2007-06-21 Institute Of Biotechnology, The Academy Of Military Medical Sciences, Pla Peptides inhibiteurs de la fusion du vih et leur utilisation
JP2009525051A (ja) * 2006-02-02 2009-07-09 トリメリス，インコーポレーテッド 改善された生物学的特性を有するｈｉｖ融合阻害剤ペプチド
EP1989220A4 (fr) * 2006-02-02 2009-05-06 Trimeris Inc Peptides inhibiteurs de fusion du vih à propriétés biologiques améliorées
EP1989220A2 (fr) * 2006-02-02 2008-11-12 Trimeris, Inc. Peptides inhibiteurs de fusion du vih à propriétés biologiques améliorées
US8034899B2 (en) 2006-02-02 2011-10-11 Trimeris, Inc. HIV fusion inhibitor peptides with improved biological properties
JP4682251B2 (ja) * 2006-02-02 2011-05-11 トリメリス，インコーポレーテッド 改善された生物学的特性を有するｈｉｖ融合阻害剤ペプチド
WO2007097903A2 (fr) 2006-02-02 2007-08-30 Trimeris, Inc. Peptides inhibiteurs de fusion du vih à propriétés biologiques améliorées
EP2045324A1 (fr) * 2006-07-27 2009-04-08 Gekkeikan Sake Co., Ltd. Procédés de production du peptide de liaison à n36
EP2045324A4 (fr) * 2006-07-27 2010-07-21 Gekkeikan Kk Procédés de production du peptide de liaison à n36
WO2008019817A1 (fr) 2006-08-17 2008-02-21 F. Hoffmann-La Roche Ag Conjugué d'un anticorps dirigé contre le ccr5 et peptide antifusiogène
US7951920B2 (en) 2006-08-17 2011-05-31 Roche Palo Alto Llc Conjugate of an antibody against CCR5 and an antifusogenic peptide
WO2008110332A1 (fr) 2007-03-13 2008-09-18 F.Hoffmann-La Roche Ag Conjugués de complément peptidique
CN101616929B (zh) * 2007-03-13 2013-08-14 弗·哈夫曼-拉罗切有限公司 肽-补体缀合物
WO2009012944A1 (fr) 2007-07-20 2009-01-29 F.Hoffmann-La Roche Ag Conjugué d'un anticorps anti-cd4 et peptides antifusogéniques
EP2526972A1 (fr) 2007-07-20 2012-11-28 F. Hoffmann-La Roche AG Conjugué d'un anticorps contre CD4 et de peptides antifusiogenes.
WO2009155789A1 (fr) * 2008-06-25 2009-12-30 中国人民解放军军事医学科学院毒物药物研究所 Polypeptides et leurs dérivés inhibant l’infection par le vih
WO2010089129A1 (fr) 2009-02-06 2010-08-12 Cormus Srl Inhibiteurs de fusion virale et leurs utilisations
WO2012020108A2 (fr) 2010-08-11 2012-02-16 Cormus Srl Inhibiteurs multimères de fusion virale et ses utilisations
WO2012035034A1 (fr) 2010-09-14 2012-03-22 F. Hoffmann-La Roche Ag Polypeptide de fusion à serpine-doigt de zinc
US9403896B2 (en) 2010-09-14 2016-08-02 Hoffmann-La Roche Inc. Serpin-finger fusion polypeptide

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JP2007517522A (ja) 2007-07-05
IL176721A0 (en) 2006-10-31
US20060247416A1 (en) 2006-11-02
BRPI0418304A (pt) 2007-05-02
RU2006128593A (ru) 2008-02-20
EP1708734A1 (fr) 2006-10-11
EP1708734A4 (fr) 2009-06-17
KR20070006708A (ko) 2007-01-11
NO20063556L (no) 2006-10-06
CA2551082A1 (fr) 2005-07-28
AU2004313242A1 (en) 2005-07-28
CN1901931A (zh) 2007-01-24

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